Dengue virus, a member of the Flaviviridae family, is a Category A pathogen that causes the most prevalent arthropod-borne viral illnesses in humans. The viral genome is an RNA molecule that plays multiple roles during viral replication. It serves as mRNA for translation, a template for RNA amplification, and substrate for encapsidation. The encapsidation process is essential for particle morphogenesis, and involves the interaction of the capsid protein with the viral genome. The flavivirus capsid is a highly basic protein that binds any nucleic acid, yet only the viral genome is encapsidated. During infection, the viral capsid protein distributes in different compartments of the infected cell. It is synthesized associated to the ER membrane and then accumulates in nucleolus and on the surface of lipid droplets. However, it is still unclear how and where the capsid protein recruits the viral RNA to produce the nucleocapsid particle. In this proposal, we will combine molecular biology, biochemistry, and genetic approaches with classical virology studies to define the mechanism of dengue virus encapsidation and its interplay with the host cell. In Aim 1, we will use novel genetic tools to dissect capsid protein requirements for particle formation and infectivity. In addition, we will define amino acids and domains of the capsid protein involved in nucleic acid binding and subcellular localization. In Aim 2, we will use cell imaging analysis together with biochemical fractionation of infected cells to define the location of nucleocapsid particle formation and the traffic of the protein between the ER, nucleolus, and lipid droplets. The link between pathogen replication and lipid droplet metabolism is an emerging topic for viruses and other infectious agents. We have recently reported that dengue virus infection induces the formation of lipid droplets, which is necessary for viral particle formation. In Aim 3, we propose to investigate how viral infection alters the protein composition of lipid droplets by proteomic studies of infected and uninfected cells. These studies will provide valuable information about host factors involved in viral replication and possible new targets for intervention. In Aim 4, we will investigate how the capsid protein modulates the genome structure using high throughput probing analysis of the viral RNA inside the virion. Dissecting the multiple functions and interactions of the capsid protein with host and viral components will shed light on a fundamental aspect of dengue and other flavivirus replication. Importantly, the studies proposed will provide new information about a viral process that has been unexplored for antiviral intervention.